FcyrIII signaling inhibitor assay

ABSTRACT

The present invention provides an assay and its use for identifying agents that inhibit the FcγRIII signaling.

The present invention relates to an assay and its use for identifying anagent that inhibits the FcγRIII signaling using a stably transfectedcell line.

Recent data with complement- and FcγR-knock out mice resulted in aparadigm shift in the understanding of the etiology of certainautoimmune diseases such as e.g. Lupus, Glomerulonephritis. While up tonow many of the autoimmune diseases were regarded to be in part or fullydependent on the complement system, these recent findings providedconclusive evidence that unwanted triggering/signaling initiated atFcγRs and in particular the FcγRIII are causally involved in thepathogenesis. Therefore the signaling cascades and their participatingmolecules may comprise novel targets for pharmacological approaches inthe prevention of autoimmune diseases, such as Lupus andGlomerulonephritis, and other diseases.

Fc receptors are expressed on a variety of cell types includingmacrophages, mast cells, neutrophils and natural killer cells.Triggering by cross-linking of Fc receptors with antibodies orantibody-antigen complexes mediates a wide variety of cellular effectorfunctions, commonly providing a link between the innate and adaptiveimmune system. The pathological importance of these systems wasdemonstrated recently by the unexpected finding that mice deficient inFc receptors were unable to mount inflammatory responses whenimmunoglobulins (IgG) are bound to their cognate antigens (see e.g.Ravetch, J. V. 1997, Fc receptors, Curr. Opin. Immunol. 9:121-125). Inmarked contrast animals deficient in complement components had a normalinflammatory response to these experimentally induced (cytotoxic)antibodies and IgG-antigen complexes, abolishing an old immunologicaldogma about the pathogenesis of many autoimmune diseases.

Pathogenic IgG antibodies arise in autoimmune diseases, includingsystemic lupus erythematosus, idiopathic thrombocytopenia purpura,immune hemolytic anemia, rheumatoid arthritis and allergic asthma andare furthermore (alone or in combination with further factors)considered as the pathogenic triggers for such diseases. In addition, itwas shown experimentally that mice deficient in IgG Fc receptors areprotected from disease in models of systemic lupus, glomerulonephritis,idiopathic thrombocytopenia purpura, hemolytic anemia and allergicasthma. It is now firmly established that the FcγRIII receptor inparticular plays an essential role in such disease processes. Suchobservations form the basis for an entirely new approach to thetreatment of intractable antibody mediated autoimmune diseases all ofthem being currently treated with IVIG (IntraVenous Immunoglobulin G).While detailed understanding of the mechanism of action of IVIG is stillunder active investigation, it is conceivable that the high amounts ofIVIG may interact with cellular FcRs and specifically with the FcγRIIIthereby preventing activation of the cells. The specificity of theFcγRIII pathway in coupling cytotoxic and immune complex antibodies toeffector responses strongly suggests that an FcγRIII signaling inhibitorwill have a similar activity comparable with IVIG with all theadvantages of a low molecular weight compound.

We have now surprisingly found and validated a stable, geneticallymodified cell line which is especially useful for high throughputscreening of inhibitors (esp. low molecular weight inhibitors) of theFcγRIII signaling after triggering with an IgG stimulus, e.g. a stimuluswith IgG complexes.

The present invention provides in one aspect an assay for identifying anagent that inhibits the FcγIII signaling comprising:

-   a) providing a cell line stably transfected with a construct    comprising an inducible specific promoter, corresponding 3′    regulatory gene sequences and a reporter gene, operatively linked to    each other,-   b) providing a candidate compound,-   c) contacting the cell line of a) with an immunoglobulin G complex    for a sufficient period of time to obtain a stimulated cell line,-   d) adding the candidate compound of b) to the cell line of a) or to    the stimulated cell line of c) simultaneously with the    immunoglobulin G complex or shortly thereafter, or not adding the    candidate compound of b),-   e) detecting the expression of the reporter gene and/or its product    in the presence and in the absence of a candidate compound and    determining whether there is a difference in the expressed amount of    the reporter gene and/or its product in the absence and in the    presence of a candidate compound,-   f) choosing an agent from said candidate compound detected in e),    e.g. for use as a pharmaceutical.

Appropriate cell lines in an assay of the present invention include anycell line where the FcγRIII signaling pathway can be triggered via animmunoglobulin G complex. Such stably transfected cell lines includee.g. a DC18 mouse cell line stably transfected with an induciblespecific promoter, corresponding 3′ regulatory gene sequences and areporter gene, operatively linked to each other.

An appropriate inducible specific promoter includes any DNA regulatorysequence that responds positively to a triggering of the FcγRIII andwhich is linked to an appropriate detectable system, e.g. a reportergene. The inducible promoter is e.g. an hTNFα promoter, thecorresponding 3′ regulatory gene sequence is e.g. from the hTNFα gene.

An appropriate reporter gene includes genes, the amount of expression ofwhich are detectable, e.g. by a method as conventional, e.g. luciferasegene, secreted alkaline phosphatase or fluorescence proteins, like e.g.GFP (=green fluorescence protein).

The cell line used in an assay according to the present invention ischaracterized in that it expresses FcγRIII at mRNA level and stronglyresponds with TNF-α secretion when stimulated with an appropriatesubstance (stimulus), e.g. with IgG complexes.

In one embodiment of the present invention the candidate compound is notadded to the cell line stimulated or not stimulated with theimmunoglobulin G complex.

In another embodiment of the present invention the screening assay canbe carried out in that the stably transfected cell line ispre-stimulated with e.g. an IgG-complex and the candidate compound isadded after a certain time period, e.g. shortly thereafter, so that thecandidate compound still can elicit its inhibitory function. Theexpression of the reporter gene and/or its product in the presence andin the absence of the candidate compound is detected as describedearlier, the difference determined and an agent may be chosen.

Appropriate candidate compounds include compound(s)(libraries) fromwhich its influence on the Fc-receptor can be determined. Compound(libraries) include for example oligopeptides, polypeptides, proteins,antibodies, mimetics, small molecules, e.g. low molecular weightcompounds (LMW's).

Since the cell lines used in the present invention strongly respond withTNF-α secretion when stimulated with appropriate substances, e.g. withIgG complexes, or to a reduction of TNF-α secretion in case thecandidate compound has an inhibitory action on the FcγRIII signalingpathway (=agent), an appropriate agent can be identified in using thesecells in an assay according to the present invention.

When TNF-α secretion is reduced also less reporter gene and/or geneproduct is expressed. The specific decrease in the expression of thereporter gene in the cell line contacted with the candidate compound inthe given concentration in comparison with the expression of thereporter gene in the stimulated cell not contacted with the candidatecompound indicates that and to what degree the candidate compoundinhibits FcγRIII signaling.

The expressed amount of the reporter gene and/or its product isdetected. A “gene product” is e.g. the expressed protein, polypeptide ora fragment thereof.

An agent is a compound which influences (inhibits) the expression of thereporter gene and/or its products detected/determined in step e). Anagent identified by an assay of the present invention is therein definedas “an agent of the present invention”.

An agent of the present invention is one of the chosen candidatecompounds and may include oligopeptides, polypeptides, proteins,antibodies, mimetics, small molecules, e.g. low molecular weightcompounds (LMW's).

Preferably an agent of the present is a candidate compound which is ableto decrease the amount of expression of the reporter gene e.g. by 35% ormore in comparison with the amount of reporter gene expressed by thecell line in the absence of a candidate compound.

The stimulation or triggering of the cell line may be of importance forthe sensitivity of the assay (ratio stimulated vs. non-stimulated). In apreferred embodiment an IgG-F(ab′)₂ complex and more preferred anIgG2a-F(ab′)₂ complex may be used as the stimulating immunoglobulin G(IgG)-antigen complex.

Further parameters like e.g. time for contacting, stimulation, number ofcells required, solvent used may be optimized according, e.g.analogously, to a method as conventional.

In another aspect the present invention provides a kit comprising

-   a) a cell line stably transfected with a construct comprising an    inducible specific promoter, corresponding 3′ regulatory gene    sequences and a reporter gene, operatively linked to each other,-   b) an immunoglobulin G-antigen complex and-   c) detection means.

Components a) and b) may be provided in appropriate solvents and/orbuffer solutions. Appropriate detection means include conventionalmeans, e.g. include fluorescence spectroscopy or ELISA techniques asconventional.

In another aspect the present invention provides an agent which inhibitsFcγRIII signaling and which is identified by the assay according to thepresent invention for use as a pharmaceutical.

An agent of the present invention may exhibit pharmacological activityand is therefore useful as a pharmaceutical, e.g. against autoimmunerelated diseases including but not limited to ITP (=idiopathicthrombocytopenia purpura), systemic lupus erythematosus, immunehemolytic anemia or rheumatoid arthritis or allergic diseases like e.g.allergic asthma or type I allergies.

An agent of the present invention may show therapeutic activity againstautoimmune related diseases and allergic diseases.

An agent of the present invention for treatment includes one or more,preferably one, agent of the present invention, e.g. a combination oftwo or more agents of the present invention.

In another aspect the present invention provides an agent for use as apharmaceutical.

In another aspect the present invention provides the use of an agent ofthe present invention for the manufacture of a medicament, e.g. apharmaceutical composition, for the treatment of autoimmune relateddiseases including but not limited to ITP (=idiopathic thrombocytopeniapurpura), systemic lupus erythematosus, immune hemolytic anemia orrheumatoid arthritis or allergic diseases like e.g. allergic asthma ortype I allergies.

In a further aspect the present invention provides a method of treatmentof autoimmune related diseases and allergic diseases, which treatmentcomprises administering to a subject in need of such treatment aneffective amount of a compound of the present invention; e.g. in theform of a pharmaceutical composition.

Treatment includes treatment and prophylaxis.

For such treatment, the appropriate dosage will, of course, varydepending upon, for example, the chemical nature and the pharmacokineticdata of an agent of the present invention employed, the individual host,the mode of administration and the nature and severity of the conditionsbeing treated. However, in general, for satisfactory results in largermammals, for example humans, an indicated daily dosage is in the rangefrom about 0.01 g to about 1.0 g, of an agent of the present invention;conveniently administered, for example, in divided doses up to fourtimes a day.

An agent of the present invention may be administered by anyconventional route, for example enterally, e.g. including nasal, buccal,rectal, oral administration; parenterally, e.g. including intravenous,intramuscular, subcutanous administration; or topically; e.g. includingepicutaneous, intranasal, intratracheal administration; e.g. in form ofcoated or uncoated tablets, capsules, injectable solutions orsuspensions, e.g. in the form of ampoules, vials, in the form of creams,gels, pastes, inhaler powder, foams, tinctures, lip sticks, drops,sprays, or in the form of suppositories.

An agent of the present invention may be administered in the form of apharmaceutically acceptable salt, e.g. an acid addition salt or metalsalt; or in free form; optionally in the form of a solvate. An agent ofthe present invention in the form of a salt may exhibit the same orderof activity as an agent of the present invention in free form;optionally in the form of a solvate.

An agent of the present invention may be used for pharmaceuticaltreatment according to the present invention alone, or in combinationwith one or more other pharmaceutically active agents.

Combinations include fixed combinations, in which two or morepharmaceutically active agents are in the same formulation; kits, inwhich two or more pharmaceutically active agents in separateformulations are sold in the same package, e.g. with instruction forco-administration; and free combinations in which the pharmaceuticallyactive agents are packaged separately, but instruction for simultaneousor sequential administration are given.

In another aspect the present invention provides a pharmaceuticalcomposition comprising an agent of the present invention in associationwith at least one pharmaceutical excipient, e.g. appropriate carrierand/or diluent, e.g. including fillers, binders, disintegrators, flowconditioners, lubricants, sugars and sweeteners, fragrances,preservatives, stabilizers, wetting agents and/or emulsifiers,solubilizers, salts for regulating osmotic pressure and/or buffers.

In another aspect the present invention provides a pharmaceuticalcomposition according to the present invention, further comprisinganother pharmaceutically active agent.

Such compositions may be manufactured according, e.g. analogously to amethod as conventional, e.g. by mixing, granulating, coating, dissolvingor lyophilizing processes. Unit dosage forms may contain, for example,from about 0.5 mg to about 1000 mg, such as 1 mg to about 500 mg.

DESCRIPTION OF THE FIGURES

FIG. 1: Stimulation of DC18 C10 cells by various immunoglobulincomplexes (including IgG isotypes); nst=non stimulated;

FIG. 2: Time kinetics for stably transfected DC18 C10 cells stimulatedwith IgG2a complexes

FIG. 3: Application of various inhibitors in the given concentration(x-axis) on IgG2a-complex stimulated DC18 C10 cells

FIG. 4: Titration of the cell number (indicated at the x-axis) toprovide for a robust readout

In the following examples all temperatures are given in degree Celciusand are uncorrected.

The following ABBREVIATIONS are used:

-   CsA=Cyclosporin A-   DNP-BSA=dinitrophenyl-bovine serum albumin-   FCS=fetal calf serum-   HEPES=(N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid))-   mGM-CSF=mouse granulocyte monocyte-colony stimulating factor-   PMA=phorbol-12-Myristate-13-acetate-   RPMI=cell culture medium named after the Roswell Park Memorial    Institute-   TNP=trinitrophenyl

EXAMPLE 1

Production of Transfected Cell Lines and Cell Cultivation:

The DC18 cell line is characterized in e.g. Elbe A. et al., J.Immunol.153:2878-2889, 1994; Prieschl E. E. et al., J.Immunol.157:2645-2653, 1996.

In short, cells are isolated/grown out from mouse fetal skin cellsuspensions, containing dermal and epidermal cells after enrichment forviable cells using a Lymphocyte-M gradient. Cells are seeded at adensity of 2×10⁵ per well in 96-well round bottom tissue culture platesusing RPMI 1640. To the medium 10% FCS, 25 mM HEPES, 50 μg/ml ofgentamycin, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mMsodium pyruvate, 50 μM β-mercaptoethanol and an antibiotio-antimycoticsolution are added. The culture medium is additionally supplemented withmGM-CSF (100 U/ml).

DC18 clone—genetically engineered cells are generated as follows:

A hTNFαpromoter—luciferase—hTNFα 3′ construct is used for generatingstable cell lines.

Cells are transfected by electroporation at optimised conditions (200V,960 pF) with a TNFα promoter—luciferase—TNFα 3′ end construct and platedin 10 ml of medium immediately after electroporation and purified byLymphoprep the next day. 1×10⁴ viable cells per well are plated in 96well microtiter plates in 100 μl of medium each plus 250 μg/ml ofgeneticin (G418). Two days later 100 μl of fresh medium are added. Everysecond day 100 μl of medium are removed and substituted by 100 μl offresh medium. Stable clones are selected by conventional cell culturemethods and single cell cloning. This results in 7 outgrowing clones ofwhich 4 respond with the induction of luciferase upon stimulation withIgG complexes to a variable degree (between 6 to 10 fold). The bestresponder is subjected to one round of further limited dilution cloning(single cell cloning) and named DC18 clone10 (=DC18 C10). This clone isselected for all further studies.

Since the stimulation of the assay is via IgG, it is of vital importancethat the cells are cultured in low IgG medium to prevent priorstimulation during the culturing period.

EXAMPLE 2

Stimulation Conditions and Readout:

IgG subtypes are provided from Pharmingen/Becton Dickinson, DNP-BSA fromCalbiochem and rabbit anti mouse Ig F(ab′)₂ fragment from JacksonLaboratories.

1×10⁴ cells are seeded in 50 μl medium per well (Optiplate, Packard).Incubation with candidate compounds is done for 45 minutes prior tostimulation with 5 μg/ml of murine IgG of different subtypes complexedeither with 10 μg/ml of DNP-BSA or 30 μg/ml of rabbit anti mouse IgF(ab′)₂ fragments. Stimulation is carried on for further 4 hours.Subsequently 13 μl of a 5 times concentrated reporter lysis buffer(Promega) are added and cells are lysed by freeze-(−80°) thaw cycle. Forthe readout 50 μl of luciferase substrate (Promega) are added andluciferase activity is measured in a MicroBetaJet (Wallac).

First experiments with this clone prove that complex formation by IgGplus F(ab′)₂ is superior to complex formation by IgG plus antigen(DNP-BSA) with respect to maximally induced values of luciferase andTNFα in the supernatant as well as overall reproducibility.

Furthermore, monomeric IgG does not stimulate TNFα induction, inagreement with no detectable expression of the high affinity receptorfor IgG, the FcγRI. Therefore, in all further experiments a stimulationby IgG plus F(ab′)₂ is performed.

As the FcγRIII in mouse and humans interacts differently with differentIgG isotypes, stimulation of DC18 C10 cells by complexes of IgG isotypesare tested instead of whole IgG, namely IgG1, IgG2a, IgG2b and IgG3complexes. As a comparison, a PMA stimulation as well as a stimulationwith IgM and IgE is done side by side. As shown in FIG. 1, the beststimulation (for IgGs) is achieved with IgG2a, which is used in allfurther studies as the triggering immunoglobulin.

To determine optimal readout conditions, time kinetics of stimulationare performed over an 6 hours range. As can be seen in FIG. 2, maximalstimulation is achieved at the 6 hours time-point. As the rationon-stimulated to stimulated, however, is already more than 10-fold at 4hours, such stimulation for the shorter time period of 4 hours ispreferred for practical reasons (to complete the assay in one dayincluding pre-incubation with drugs and measurement) for all furtherstudies,.

EXAMPLE 3

Screening for Prototype Inhibitors:

In search for a prototype inhibitor (positive control) several candidatecompounds known to inhibit certain signaling pathways are exploited.Neither CsA (Sandimmune; data not shown), FK506 (FIG. 3A) or Wortmannin(a PI3-kinase inhibitor; FIG. 3B) inhibit the readout significantly atIC₅₀ concentrations. Quite in contrast, Apigenin, a commerciallyavailable erk1,2/jnk1,2 inhibitor, inhibits IgG2a complexes-provokedluciferase induction at the described IC₅₀ concentration (see FIG. 3C).

To optimize for high throughput screening a titration of the number ofcells required in order to obtain a suitable/significant luciferasereadout is carried out. As can be seen in FIG. 4, 10⁴ DC18 C10 cells perwell in 96 well microtiter plate are sufficient for a robust andeasy-to-detect readout.

EXAMPLE 4

Establishment of an Assay Protocol:

An assay protocol is established as follows.

-   1.) 10⁴ DC18 C10 cells per well are used in 45 μl of medium (Packard    Optiplate) and 5 μl of compounds in solution/solvent control are    added,-   2.) Incubation is carried out for 45 minutes at 37° in a humidified    incubator by 5% CO₂,-   3.) Stimulation is carried out with 5 μg/ml of mouse IgG2a anti-TNP    plus 30 μg/ml of rabbit anti-mouse Ig(Fab′)₂ fragment,-   4.) Incubation is carried out for 4 hours at 37° in a humidified    incubator by 5% CO₂,-   5.) 13 μl of a 5 times concentrated lysis buffer (Promega) are    added,-   6.) the composition is frozen for 1 hour at −80° , cells are thawed    again so that they are lysed (lysis by freeze thaw cycle),-   7.) 50 μl of luciferase reagent (Promega) are added to the lysed    cells and with 5 seconds lag time, 5 seconds measuring time in a    Wallac MicroBetaJet measurements of luciferase activity are done.

The cut off point for the definition of a hit is set at 35% inhibitionat 5 μM concentration of substance, but can be chosen also differently,e.g. at 60% inhibition.

The whole procedure including handling the cells and measuring can bedone in a six to seven hours time-span, making the assay easy to handlefor high throughput screening. Using this assay protocol we alsovalidated the assay by checking for high/low values, solvent sensitivityand performed a number of test racks with various substances.

1. An assay for identifying an agent that inhibits the FcγRIII signalingcomprising: a) providing a cell line stably transfected with a constructcomprising an inducible specific promoter, corresponding 3′ regulatorygene sequences and a reporter gene, operatively linked to each other, b)providing a candidate compound, c) contacting the cell line of a) withan immunoglobulin G complex for a sufficient period of time to obtain astimulated cell line, d) adding the candidate compound of b) to the cellline of a) or to the stimulated cell line of c) simultaneously with theimmunoglobulin G complex or shortly thereafter, or not adding thecandidate compound of b), e) detecting the expression of the reportergene and/or its product in the presence and in the absence of acandidate compound and determining whether there is a difference in theexpressed amount of the reporter gene and/or its product in the absenceand in the presence of a candidate compound, f) choosing an agent fromsaid candidate compound detected in e), e.g. for use as apharmaceutical.
 2. Assay according to claim 1, characterized in that theimmunoglobulin G (IgG)-antigen complex is an IgG-F(ab′)₂ complex.
 3. Akit comprising a) a cell line stably transfected with a constructcomprising an inducible specific promoter, corresponding 3′ regulatorygene sequences and a reporter gene, operatively linked to each other, b)an immunoglobulin G-antigen complex, and c) detection means.
 4. An agentidentified by an assay according to claim
 1. 5. An agent of claim 4 foruse as a pharmaceutical.
 6. The use of an agent of claim 4 for themanufacture of a medicament for the treatment of an autoimmune relateddisease or an allergic disease.
 7. A pharmaceutical compositioncomprising an agent identified by an assay according to claim 1 inassociation with at least one pharmaceutical excipient.
 8. A method oftreatment of an autoimmune related disease or an allergic disease, whichtreatment comprises administering to a subject in need of such treatmentan effective amount of an agent identified by an assay according toclaim 1 or a pharmaceutical composition comprising an agent identifiedby an assay according to claim 1 in association with at least onepharmaceutical excipient.
 9. A method according to claim 8 characterizedin that the autoimmune related disease is idiopathic thrombocytopeniapurpura, systemic lupus erythematosus, immune hemolytic anemia orrheumatid arthritis and the allergic disease is allergic asthma.